Gas ChromatographyGas Chromatography

Mobile phase:Mobile phase: Inert gas such as N2 or He.Inert gas such as N2 or He.

Stationary phase:Stationary phase: May beMay be solid (GSC) or solid (GSC) or

Non volatile liquid at operating temperature Non volatile liquid at operating temperature over inert support (GLC).over inert support (GLC).

Mixture:Mixture: MixtureMixture componentscomponents must be:must be:

11- - Volatile: Volatile material usually Volatile: Volatile material usually have molecular weight not exceed 400 – 500 have molecular weight not exceed 400 – 500 Dalton.Dalton.

22- - Thermo stable. Thermo stable.

Instruments: Instruments:

1- Gas Tank.1- Gas Tank.

2- Flow meter.2- Flow meter.

3- Injector system for sample 3- Injector system for sample application.application.

4- Column and oven.4- Column and oven.

5- Detector.5- Detector.

6- Amplifier and Recorder.6- Amplifier and Recorder.

stationary phase:stationary phase:

GSC:GSC: 1- Silica gel.1- Silica gel.

2- Charcoal (Activated). 2- Charcoal (Activated).

3- Molecular sieves (synthetic inorganic 3- Molecular sieves (synthetic inorganic materials).materials).

GLC:GLC:

Liquid or low – melting solid coating an inert Liquid or low – melting solid coating an inert support.support.

e.g. of supports:e.g. of supports:

11- - Chromosorb.Chromosorb.

22- - Celite.Celite.

33- - Glass beedsGlass beeds..

44- - Teflon Teflon

e.g. of liquids or low-melting solids:e.g. of liquids or low-melting solids:

Each liquid have aEach liquid have a maximum operating maximum operating Temperature.Temperature.

Silicon gum rubber (higher than 250 ˚C).Silicon gum rubber (higher than 250 ˚C). Paraffin oil (250 ˚C).Paraffin oil (250 ˚C). Polyethylene glycol (up to 250 ˚C).Polyethylene glycol (up to 250 ˚C).

Column:Column:

1- Packed Columns:1- Packed Columns: 2-6 mm id and 1-3 2-6 mm id and 1-3 meters in length.meters in length.

2- Capillary Column:2- Capillary Column: 0.2 – 0.5 mm id 0.2 – 0.5 mm id and 10-100 meters in length. Some columns and 10-100 meters in length. Some columns may reach up to 300 meters.may reach up to 300 meters.

Types of capillary columns:Types of capillary columns:1-1-Wall Coated Open tubular (WCOT):Wall Coated Open tubular (WCOT):Used in GLC, the stationary liquid coats the column wall.Used in GLC, the stationary liquid coats the column wall.

2-Support Coated Open tubular (SCOT):2-Support Coated Open tubular (SCOT):The inert support is fused to the column wall by special The inert support is fused to the column wall by special

machine then the stationary liquid coat the support. It is machine then the stationary liquid coat the support. It is used in GLC.used in GLC.

3-Porous layer Open tubular (PLOT):3-Porous layer Open tubular (PLOT):The stationary solid is bonded to the wall of the column. The stationary solid is bonded to the wall of the column. Not commercially produced.Not commercially produced.

4-Micro-Packed Columns:4-Micro-Packed Columns:The columns are coiled and packed with the desired The columns are coiled and packed with the desired

support with stationary liquid or solid stationary phase support with stationary liquid or solid stationary phase under pressure and vibrations.under pressure and vibrations.

Capillary Columns give mush better resolution but their sample Capillary Columns give mush better resolution but their sample capacity is much lower than the packed columns (about 1/10 or capacity is much lower than the packed columns (about 1/10 or less). less).

Liquid film(Sationary Phase)

SupportSolid Staionary Phase

Micro-Packed PLOT SCOT WCOT

Operating Temperature:Operating Temperature:Operating Temperature can be looked at as the Operating Temperature can be looked at as the polarity of mobile phase in case of adsorption polarity of mobile phase in case of adsorption chromatography. chromatography.

Very low temp: Tailing of peaks.Very low temp: Tailing of peaks.

Very high temp: No separation of peaks.Very high temp: No separation of peaks. Temperature programming:Temperature programming:

Allows starting the process at low temperature and Allows starting the process at low temperature and gradually increasing the temperature till reach the gradually increasing the temperature till reach the maximum required temperature.maximum required temperature.

Column bleeding:Column bleeding:Elution of the liquid stationary phase itself if very high Elution of the liquid stationary phase itself if very high temperature is used (exceed maximum operating temperature is used (exceed maximum operating temperature).temperature).

Conditioning of Columns:Conditioning of Columns:

Means heating the column before use at a Means heating the column before use at a temperature 25-50 0C above the normal temperature 25-50 0C above the normal working temperature. However, that must not working temperature. However, that must not exceed the maximum operating temperature. exceed the maximum operating temperature. The mobile phase is allowed to flow during the The mobile phase is allowed to flow during the conditioning but the column outlet must be conditioning but the column outlet must be disconnected from the detector. Conditioning disconnected from the detector. Conditioning aims to clean the column from any impurities.aims to clean the column from any impurities.

Detectors:Detectors:

They are used to indicate the presence They are used to indicate the presence and measure the amount of the and measure the amount of the components in the column effluent.components in the column effluent.

Examples of the commonly used Examples of the commonly used detectors are:detectors are:

11- - Thermal Conductivity Detector Thermal Conductivity Detector (TCD). (TCD).

22- - Flame Ionization Detector (FID). Flame Ionization Detector (FID).

33-- Electron Capture Detector (ECD). Electron Capture Detector (ECD).

Sample IntroductionSample Introduction

Sample introduction will affect the separation, Sample introduction will affect the separation, accuracy and precision of the results. The accuracy and precision of the results. The method of sample application will depends method of sample application will depends mainly on the nature of the sample.mainly on the nature of the sample.

Gaseous samples:Gaseous samples:

They are introduced using special valves.They are introduced using special valves.

Liquid Samples:Liquid Samples:Include liquids, liquids or solids in suitable Include liquids, liquids or solids in suitable solvents. They are introduced by injection via solvents. They are introduced by injection via septum seal.septum seal.

Capillary column have low capacity so we have Capillary column have low capacity so we have to apply very small amount of samples. This can to apply very small amount of samples. This can be achieved by:be achieved by:

1- Dilution of sample with solvent:1- Dilution of sample with solvent:

This will lead to problem due to large amount of solvents that This will lead to problem due to large amount of solvents that may affect separation and quantitation.may affect separation and quantitation.

2- Injection of very small amount of more concentrated 2- Injection of very small amount of more concentrated solution:solution:

That leads to increase the error in measurements.That leads to increase the error in measurements.

3- Using modified injectors that allow splitting of 3- Using modified injectors that allow splitting of samples: samples:

Only part of the injected sample go to the column and the Only part of the injected sample go to the column and the rest of the sample go to vent. The split ratio to give good rest of the sample go to vent. The split ratio to give good results is usually 1: 100.results is usually 1: 100.

Head Space Sampling:Head Space Sampling:

The sample is contained in sealed vial and The sample is contained in sealed vial and maintained at constant temperature for maintained at constant temperature for about 20 min. so equilibrium will be about 20 min. so equilibrium will be achieved between gas and liquid states. achieved between gas and liquid states. The vapour phase is then allowed to enter The vapour phase is then allowed to enter the column.the column.

Derivatization of non volatile Derivatization of non volatile samples:samples:

Non volatile samples containing acidic Non volatile samples containing acidic hydrogen can be converted into more hydrogen can be converted into more volatile more thermally stable derivatives volatile more thermally stable derivatives as:as:

1- Silylation:1- Silylation:

This is the most widely used method. This is the most widely used method. Alkylsilyl group will replace the acidic Alkylsilyl group will replace the acidic hydrogen.hydrogen.

pyridinepyridine

R-OHR-OH++Cl-SiMeCl-SiMe33 R-O-SiMeR-O-SiMe33 + + HClHCl

(TMS) (TMS)

TrimethylchlorosilaneTrimethylchlorosilane

2- Acylation:2- Acylation:

COCFCOCF33

R-OH + O R-O-COCFR-OH + O R-O-COCF33

COCFCOCF33

(TFAA)(TFAA)

TrifluoroaceticanhydrideTrifluoroaceticanhydride

3- Alkylation:3- Alkylation:

RCOOH + BFRCOOH + BF33/MeOH RCOOMe /MeOH RCOOMe

Boron trifluoride Boron trifluoride

Peak symmetry:Peak symmetry:

Good system must result in a symmetric peaks with Good system must result in a symmetric peaks with narrow base. Asymmetry ratio (As) can be narrow base. Asymmetry ratio (As) can be calculated:calculated:

bb

As = ---------- As = ----------

aa

Where a and b should by measured at10% height of the Where a and b should by measured at10% height of the peak. peak.

As for good peak will be 0.9- 1.1.As for good peak will be 0.9- 1.1.

ba 10% height of the peak

The peak shape also can be predicted by calculation The peak shape also can be predicted by calculation of the Tailing factor (T):of the Tailing factor (T):

WW T= --------T= -------- 2F2FWhere W and F must be measured at 5% height of the peak.Where W and F must be measured at 5% height of the peak.Ideal peak will give Tailing factor = 1. Increasing the value of T Ideal peak will give Tailing factor = 1. Increasing the value of T

means Tailed peak.means Tailed peak.

5% height of the peakF W

Resolution:Resolution:It is the measure of the efficiency of the separation of It is the measure of the efficiency of the separation of two components in a mixture. Resolution "R" can be two components in a mixture. Resolution "R" can be calculated:calculated:

VRa

VRb

Sam

ple

inje

ctio

n

half peak heighthalf peak

height

Wha

Whb

1.18(V1.18(VRbRb-V-VRaRa))

R = R = ---------------------- ----------------------

WWhaha + W + Whbhb

VVRaRa distance along the base line between the point of distance along the base line between the point of Injection and the maxima of a.Injection and the maxima of a.

WWhaha is the peak width measured at half peak height. is the peak width measured at half peak height.

Increase in R means better separation and absence Increase in R means better separation and absence of overlapping between peaks.of overlapping between peaks.

Identification of peaksIdentification of peaks

Retention timeRetention time ( (ttRR):):

It is the time between sample injection It is the time between sample injection and the maximum detection of a and the maximum detection of a substance under specified conditions substance under specified conditions (temperature, gas flow, Column (temperature, gas flow, Column type…………….etc).type…………….etc).

Relative Retention time (Relative Retention time (α):α):Since tR is vary due to many factor so it is hard to Since tR is vary due to many factor so it is hard to use it as a method for identification. To overcome use it as a method for identification. To overcome this problem we camper tthis problem we camper tR R of the substance with of the substance with ttS S (Retention time of a standard substance). (Retention time of a standard substance).

ttRR- t- taa

α α = = ------------ ------------

ttSS- t- taa

ttaa: Retention time of air peak.: Retention time of air peak.

The standard is mixed with the substance and The standard is mixed with the substance and injected together so conditions are the same.injected together so conditions are the same.

Retention Index (I):Retention Index (I):The identification based on the comparison of the The identification based on the comparison of the retention time of the substance not only with one retention time of the substance not only with one standard but with series of hydrocarbons (n-standard but with series of hydrocarbons (n-paraffins Cparaffins CnnHH22n+n+22). “I” can be calculated by ). “I” can be calculated by equation or from graphs or by the aid of computer equation or from graphs or by the aid of computer programs.programs.

Peak Identification by GC-MS:Peak Identification by GC-MS:The peaks elapsed from the GC are subjected to The peaks elapsed from the GC are subjected to mass spectrometer to determine their molecular mass spectrometer to determine their molecular weight and fragmentation pattern for the weight and fragmentation pattern for the identification purpose.identification purpose.